A method for detection of point mutations in target nucleic acid using loop-mediated isothermal amplification

Abstract

Various embodiments relate generally to the field of nucleic acid amplification and detection, in particular loop-mediated isothermal nucleic acid amplification and the detection of amplicons using designed detection probes. Moreover, various embodiments also relate to methods and kits for determining the presence or quantity of point mutations in a target nucleic acid molecule in a sample using loop-mediated isothermal amplification, which may be used for identifying genetic variants. In one aspect, the detection probe is a single-stranded probe comprising a nucleotide sequence fully complementary to a nucleotide sequence of the probe binding site comprising the point mutation. In another aspect, the detection probe comprising a nucleotide complementary to the point mutation at the penultimate base relative to the 3′ end of the detection probe.

1 . A method for determining the presence, or quantity, of a point mutation, preferably a single nucleotide polymorphism (SNP), in a target nucleic acid molecule in a sample using loop-mediated isothermal amplification (LAMP), the method comprising:
 (a) combining a LAMP reaction mixture, DNA polymerase with 5′→3′ polymerase activity, and a detection probe with the sample (suspected of containing the target nucleic acid molecule),   wherein the LAMP reaction mixture comprises a LAMP primer set of 4 to 6 primers, comprising two inner primers (FIP and BIP), two outer primers (F3 and B3), and optionally one or two loop primers (LF and/or LB), wherein each primer recognises a distinct primer binding site within the target nucleic acid molecule,   wherein the detection probe recognises a probe binding site within target amplicons,   wherein the detection probe is a single-stranded probe comprising:   a nucleotide sequence fully complementary to a nucleotide sequence of the probe binding site comprising the point mutation;   a nucleotide complementary to the point mutation at the penultimate base relative to the 3′ end of the detection probe; and   a locked nucleic acid (LNA) or peptide nucleic acid (PNA) residue at the first, second, third and/or fourth, preferably third, or second and third, or first and third, or third and fourth nucleotide position relative to the 3′ end of the detection probe,   wherein the detection probe comprises a quencher-fluorophore pair at opposite ends of the probe at a distance that allow the quencher to quench the fluorophore signal, wherein the detection probe can hybridize to said target amplicons under LAMP assay conditions and form a double-stranded probe: target complex,   (b) amplifying the target nucleic acid molecule by LAMP under suitable assay conditions that allow:   i. generation of the target amplicons;   ii. hybridization of the detection probe to the target amplicons to form the probe: target complex; and   iii. cleavage of the detection probe by the DNA polymerase to release the quencher or fluorophore; and   (c) detecting and optionally quantifying the released quencher or fluorophore to determine the presence or quantity of the point mutation in the target nucleic acid molecule in the sample.   
     
     
         2 . The method of  claim 1 , wherein the DNA polymerase with 5′→3′ polymerase activity is a Bst polymerase selected from Bst3 polymerase, Bst2 polymerase and IsoPol+. 
     
     
         3 . The method of  claim 1 , wherein the primer set further comprises two swarm primers. 
     
     
         4 . The method of  claim 1 , wherein the primers are designed to amplify loci in the target nucleic acid molecule comprising the point mutation relative to a reference wild-type nucleotide sequence, wherein the nucleotide sequence of the probe binding site comprises the point mutation. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the LNA or PNA residue is positioned at the third nucleotide position relative to the 3′ end of the detection probe. 
     
     
         7 . The method of  claim 1 , wherein the LNA or PNA residue is positioned at the first and third or second and third nucleotide position relative to the 3′ end of the detection probe. 
     
     
         8 . The method of  claim 1 , wherein the probe binding site is different from and non-overlapping with any one of the primer binding sites. 
     
     
         9 . The method of  claim 1 , wherein the detection probe is 14-23 nucleotide bases in length, preferably 14-21 nucleotide bases in length. 
     
     
         10 . The method of  claim 1 , wherein the detection probe comprises a phosphorothioate bond at the 3′-end. 
     
     
         11 . The method of  claim 1 , wherein the detection probe comprises at least one additional modified nucleotide residue to either increase the detection probes melting temperature (Tm) or binding affinity. 
     
     
         12 . The  method of 1 , wherein the fully complementary double-stranded probe: target complex has a higher melting temperature (Tm) in comparison to a non-fully complementary double-stranded probe: target complex in which the target nucleic acid comprises at least one mismatched nucleotide. 
     
     
         13 . The method of  claim 12 , wherein the melting temperature (Tm) of the fully complementary double-stranded probe: target complex is higher by about 1.5° C. or greater in comparison to the non-fully complementary double-stranded probe: target complex. 
     
     
         14 . The method of  claim 1 , wherein the fluorophore is attached to the 5′ end of the detection probe and the quencher is attached to the 3′ end of the detection probe. 
     
     
         15 . The method of  claim 1 , wherein the method is a multiplexing method and is for determining the presence, or optionally quantity, of a point mutation in two or more target nucleic acid molecules in the sample, wherein the method uses one or more primer sets and one or more detection probes for each target nucleic acid molecule or for multiple related target nucleic acid molecules. 
     
     
         16 . The method of  claim 1 , wherein the point mutation is a biomarker for a disease or condition in a subject, optionally cancer. 
     
     
         17 . The method of  claim 1 , wherein the target nucleic acid molecule is a nucleic acid of a pathogen, optionally a human pathogen, preferably a bacterial, fungal, parasite or viral nucleic acid molecule or a cDNA reverse transcript of a bacterial, fungal, parasite or viral RNA. 
     
     
         18 . The method of  claim 17 , wherein the pathogen is a coronavirus, influenza virus, paramyxovirus or enterovirus. 
     
     
         19 . The method of  claim 18 , wherein the target nucleic acid molecule is a nucleic acid of SARS-COV-2 virus, optionally variants of the SARS-COV-2 virus selected from alpha, beta, gamma and delta variants, optionally wherein the nucleic acid of SARS-CoV-2 virus is an S-gene comprising a point mutation relative to a reference wild-type S-gene nucleotide sequence. 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein the sample is not subjected to a purification step prior to step (a). 
     
     
         22 . (canceled) 
     
     
         23 . A kit for determining the presence, or quantity, of a point mutation, preferably single nucleotide polymorphism (SNP), in a target nucleic acid molecule in a sample using loop mediated isothermal amplification (LAMP), the kit comprising:
 a LAMP reaction mixture comprises a LAMP primer set of 4 to 6 primers, comprising two inner primers (FIP and BIP), two outer primers (F3 and B3), and optionally one or two loop primers (LF and/or LB), wherein each primer recognises a distinct primer binding site within the target nucleic acid molecule;   a DNA polymerase with 5′→3′ polymerase activity; and   a detection probe that recognises a probe binding site within target amplicons, wherein the detection probe is a single-stranded probe comprising:   a nucleotide sequence fully complementary to a nucleotide sequence of the probe binding site comprising the point mutation;   a nucleotide complementary to the point mutation at the penultimate base relative to the 3′ end of the detection probe; and   a locked nucleic acid (LNA) or peptide nucleic acid (PNA) residue at the first, second, third and/or fourth, preferably third, or second and third, or first and third, or third and fourth nucleotide position relative to the 3′ end of the detection probe,   wherein the detection probe comprises a quencher-fluorophore pair at opposite ends of the probe at a distance that allows the quencher to quench the fluorophore signal, wherein the detection probe can hybridize to said target amplicons under LAMP assay conditions and form a double-stranded probe: target complex.